The present invention concerns a new method of checking the performance of a flow cytometer, the flow cytometer comprising means delivering electrical pulse signals indicating the presence of particles, such as somatic cells or bacteria. The invention is specifically dedicated to milk testing and more specifically to determination of the number of somatic cells in milk. Further according to the present invention the method allows for inspection and correction for any agglomeration of particles in a standard sample used for checking the performance of the flow cytometer. Further the invention concerns an apparatus comprising data processing equipment arranged to execute the above method, as well as a stannard kit for application in the apparatus.
Flow cytometers are instruments used for analysis of particles suspended in a fluid, e.g. biological cells in body fluids, such as somatic cells in milk. Briefly, a flow cytometer comprises a liquid flow system and an optical system intersecting each other in a flow cuvette, as well as an electronic system, detecting fluorescence or scatter originating from particles passing through the cuvette. Preferably, the fluid sample flows in a single, thin string inside the cuvette thereby allowing any particles present to be counted one by one. Further, in a preferred embodiment the thin string is surrounded by a sheath fluid. Thanks to the sheath fluid, casual impurities, present in the fluid and having a size larger than the particles or cells to be counted and accordingly larger than the size of the string of fluid sample, will not cause clogging. They might have done so if flowing in a liquid channel as narrow as the thin inner string.
The method according to the invention is specifically developed for flow cytometers applied for fast determination of the number of cells in a milk sample or a milk product and more specifically for the fluorescent type of flow cytometers, in which the cells or particles are all stained by a fluorescent dye, which reacts fluorescent when exposed to illumination. A fluorescence signal of adequate size, i.e. above noise, is considered to indicate the passage of a particle or cell. In the preferred embodiment of the flow cytometer, the presently preferred agent or dye is Ethidium Bromide. The present type of flow cytometer has only one channel, i.e. it is optimised to detect only one kind of fluorescence or scatter. However, flow cytometers may have a plurality of channels, each dedicated to a specific dye.
The somatic cell count is considered a measure of the milk quality (high quality milk has a low cell count). Accordingly the cell count can be applied by the dairy when setting the price according to which the farmer is paid for the delivered milk. In order to ensure correct payment to the farmer and to reveal any milk of too low quality, a proper functioning of the flow cytometer is crucial.
In order to obtain accurate and reproducible results flow cytometers must be aligned and calibrated. The light ray from the illumination source must hit the stream of particles and the detector optics must focus on the particle stream when the illuminated particles exhibit fluorescence. Also the gain and characteristics of the electronic circuits processing the detected signals and the applied mathematics must produce a number indicating the true number of particles in the stream with a sufficient accuracy and reproducibility.
Possible Reasons for Poor Performance
Any defects or maladjustment of flow rates (or clogging) in the flow system and/or defects or misalignment of the optical system may cause the count to either decrease or increase compared to the true value.
Prior Art
U.S. Pat. No. 5,093,234 (Schwartz) discloses a method of aligning, compensating and calibrating a flow cytometer for analysis of samples and a microbead standard kit therefore. The method can be applied to multi-channel flow cytometers. The method includes running test measurements on standard kits; adjusting fluorescence channel PMT voltages and gain to position resulting dot plot or histogram near the origin of the axis of each of the fluorescence channels; and setting boundary levels in each channel to indicate fluorescence intensity. The standard kit comprises a blank and/or an auto-fluorescent microbead population and at least two series of calibrated microbead populations labelled with fluorescent dye(s). U.S. Pat. No. 5,084,394 (Vogt et al) discloses a similar method for corrective calibration of a flow cytometry using a mixture of fluorescent microbeads and cells. These methods provide for an advanced, sophisticated and qualitative analysis of single cells such as lymphocytes in blood.
The present method concerns a xe2x80x9cperformance checkxe2x80x9d, i.e. a monitoring of functional performance, of a single channel flow cytometer for counting somatic cells in milk. The instrument is specifically designed for fast sample handling and counting, allowing about 500 samples pr hour to be counted. This kind of flow cytometer counts the cells based upon measuring only one parameter, such as green or red fluorescence. The instrument is not intended for qualitative studies of the cells. The performance check method is based on the use of a standard and/or calibration fluid comprising only one type of particles or microbeads which are unstained until they enter the process according to the present invention. Thereby the standard fluid samples are as simple as possible. The standard samples are very stable and adequate for a long term shelf life, i.e. a great number of standard samples may be stored by the user for months or years for future use, such as a regular performance check every morning or when ever necessary.
Besides a thorough check of the operation of the flow cytometer the standard fluid could also be applied for a calibration of the flow cytometer.
The present invention provides a method of checking the performance of a flow cytometer instrument, in which instrument the number of particles or cells in a fluid flow are counted by providing data representing a PHA diagram (Pulse Height Analysis), of registered pulses. The invention is characterised byxe2x80x94providing a lot of standard samples, including only one type of substantially uniform microbeads,xe2x80x94providing data representing an optimal (desired) PHA diagram (PHA0) of the pulses registered, when measuring a standard sample from the lot on a reference instrument, said data being stored in a memory in the instrument itself, or a memory in data processing means connected to the instrument, or in means from which the data may be imported into the instrument or into the data processing means connected to the instrument,xe2x80x94measuring a standard sample on the instrument to be checked,xe2x80x94providing data representing a PHA diagram (PHAS) for the pulses registered during the measurement of the standard sample on the instrument to be checked,xe2x80x94comparing the present PHA diagram (PHAS) to the optimal PHA diagram (PHA0),xe2x80x94and analysing and/or evaluating said data in order to determine any poor or faulty operation of the instrument. By this method the user can check the instrument regularly, and the user can readily be informed of any precautions to be taken. Preferably the microbeads in the lot are unstained until the enter the instrument. The use of unstained microbeads are specific favourable in that also the staining process in the instrument is controlled when measuring the standard sample.
Preferably at least one of the following parameters are calculated: a particle count, a plurality of particle counts on the same sample, a standard deviation, s, and/or Coefficient of Variation, CV, based on (at least two, preferably three) repeated/consecutive measurements on the same sample and substantially at the same time, a signal mean value, and a signal width, i.e. the width of the bell-curve in the PHA-diagramxe2x80x94the corresponding data for the standard sample of the standard fluid measured on a reference instrument. i.e. the optimum values of said data, being provided with the lotxe2x80x94comparing at least one of the above parameters for the actual measurement of standard sample to the corresponding optimal parameters of the standard fluid,xe2x80x94and analysing said data to estimate whether the instrument is operating substantially optimally, (i.e. within preferred limits) or is not operating substantially optimally (i.e. outside preferred limits), registering any off-limit deviations from optimal operation.
Preferably the registered off-limit deviations are considered as symptoms which are displayed to the user.
Preferably the data processing equipment comprises means for evaluating the observed symptoms, proposing possible defects (make a diagnosis), and making recommendations for how to remedy any poor performance, and/or any precautions to be taken. Preferably the means for evaluating the symptoms include a library stored in memory means arranged to be accessed by the data processing means. Preferably, the means for checking the performance/evaluating the symptoms includes display of a list of consecutive results to the user on a monitor and/or providing a print out. Preferably, the means for checking the performance/evaluating the symptoms includes display of detailed results and data of recent measurements of the standard fluid. Preferably the means for checking the performance/evaluating the symptoms includes displaying a measured PHAxe2x80x94diagram on request from the user.
Preferably the means for checking the performance/evaluating the symptoms includes display of a list of possible situations comprising defined symptoms and for each situation present information on possible reasons and appropriate actions to remedy the defect.
The display of any symptoms and actions to remedy any default operation is advantageous in that the user himself will be able to adjust the instrument to obtain optimum performance. Thereby the visit by a service engineer can be avoided. Thereby the loss of precious measurement time due to poor performance is avoided.
The apparatus according to the invention is arranged to execute the above method. The standard kit according to the invention comprises a standard fluid including a plurality of substantially uniform microbeads and associated means carrying information on the lot, and specifically on the content of microbeads in the standard fluid.
The method and kit is specifically favourable in that the test samples (standard samples) are handled in the same way as any other sample, and any defect in the instrument, which is liable to influence an ordinary measurement result will also be liable to in influence the result of the test measurement on the standard sample.
Further scope of the applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.